Oaks are a dominant and key tree species in Mediterranean forest ecosystems. However, in recent decades, oak forests have been heavily impacted by oak decline, a worldwide phenomenon exacerbated by climate change. The charcoal disease agent Biscogniauxia mediterranea is involved in the decline of Mediterranean oak formations in a variety of contexts. Here, we investigated the impact and role of B. mediterranea in decline of oaks in Castelfidardo Forest, a relict wood of the late Holocene and a Site of Community Importance. We established five plots within which we recorded the tree positions, any symptoms and signs of decline, association of B. mediterranea to declining trees, and the deadwood and associated mycota. Of 471 oaks inspected, 7.0% showed brownish exudates on the stems, 46.9% showed epicormic shoots along the main trunk, and 24.4% showed black carbonaceous stromata on diseased branches and trunks. The decline was most severe for Quercus cerris, which comprised plots #4 and #5, at 50.0% (81/162 trees) and 29.0% (33/114), respectively; then for Quercus robur for plot #3, at 40.0% (38/95); and finally for Quercus pubescens for plots #1 and #2, at 13.7% (7/51) and 12.3% (6/49), respectively. Bark tissue was collected from trees with charcoal cankers, with microscopy examination and identification by mycological and molecular methods. This investigation revealed close association between oaks with pronounced reduction of vitality and incidence of B. mediterranea. The deadwood was equally distributed among the five plots, and was heavily colonized by Basidiomycota. The high incidence of this oak-weakening pathogen B. mediterranea appears to be related to stress conditions (e.g., water scarcity, prolonged drought). However, absence of silvicultural management, high competition among physiologically mature trees, and the geographic isolation of this residual forest might have predisposed these oaks to decline.